Article pubs.acs.org/est
Species Differences Take Shape at Nanoparticles: Protein Corona Made of the Native Repertoire Assists Cellular Interaction Yuya Hayashi,*,†,‡ Teodora Miclaus,† Carsten Scavenius,†,§ Katarzyna Kwiatkowska,∥ Andrzej Sobota,∥ Péter Engelmann,⊥ Janeck J. Scott-Fordsmand,‡ Jan J. Enghild,†,§ and Duncan S. Sutherland*,† †
iNANO Interdisciplinary Nanoscience Center, Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus C, Denmark Department of Bioscience-Soil Fauna Ecology and Ecotoxicology, Aarhus University, Vejlsøvej 25, 8600 Silkeborg, Denmark § Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10, 8000 Aarhus C, Denmark ∥ Department of Cell Biology, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland ⊥ Department of Immunology and Biotechnology, Clinical Center, University of Pécs, Szigeti u. 12, Pécs H-7643, Hungary ‡
S Supporting Information *
ABSTRACT: Cells recognize the biomolecular corona around a nanoparticle, but the biological identity of the complex may be considerably different among various species. This study explores the importance of protein corona composition for nanoparticle recognition by coelomocytes of the earthworm Eisenia fetida using E. fetida coelomic proteins (EfCP) as a native repertoire and fetal bovine serum (FBS) as a non-native reference. We have profiled proteins forming the long-lived corona around silver nanoparticles (75 nm OECD reference materials) and compared the responses of coelomocytes to protein coronas preformed of EfCP or FBS. We find that over time silver nanoparticles can competitively acquire a biological identity native to the cells in situ even in non-native media, and significantly greater cellular accumulation of the nanoparticles was observed with corona complexes preformed of EfCP (p < 0.05). An EfCP-nanoparticle mimicry made with a recombinant protein, lysenin, revealed its critical contribution in the observed cell−nanoparticle response. This confirms the determinant role of the recognizable biological identity during invertebrate in vitro testing of nanoparticles. Our finding shows a case of species-specific formation of biomolecular coronas, and this suggests that the use of representative species may need careful consideration in assessing the risks associated with nanoparticles.
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INTRODUCTION Nanoparticles undergo considerable environmental transformations before reaching the target biological system,1 but their behavior and fate within the organism are then dictated by their biological identitythe biomolecular corona around nanoparticles (extensively reviewed in refs 2 and 3). Nature has diversified protein repertoires through evolution,4 and thereby the biological identity of nanoparticle−protein complexes may be considerably different among various species where there is little evolutionary conservation in the repertoire. Species differences of the nanoparticle’s biological identity could thus be translated into unique entities, each potentially having different toxicokinetics even among related species sharing similar ecological niches. Recent studies have characterized the formation of biomolecular coronas following incubation of different types of nanoparticles in fetal bovine serum (FBS) and human blood plasma (for a review see ref 2). Proteins interacting at nanoparticles may, for example, suffer conformational changes5,6 with resultant alteration of the biological functioning7,8 or initiation of inflammatory signaling.9 Nanoparticle− © 2013 American Chemical Society
protein complexes can acquire a differential biological identity and alter cell responses simply by whether the serum supplemented has been heat-inactivated,10 even with respect to the composition of cell culture media.11 In contrast, no studies exist that have explored the interaction of nanoparticles with proteins of invertebrate origins, although model invertebrate animals have been increasingly applied to in vitro settings12−14 as an integral part of nanomaterials ecotoxicology studies assessing at multilevels of biological organization.15−18 It is clear that adoption of mammalian in vitro practice creates an artificial biological milieu, in which the cells of invertebrates encounter exotic proteins of vertebrates and, most importantly, coronas of those extraneous proteins around nanoparticles. We have previously investigated fundamental aspects of how silver nanoparticles (AgNPs) bring about stress and immune responses that may be deeply conserved despite the evoluReceived: Revised: Accepted: Published: 14367
September 17, 2013 November 15, 2013 November 18, 2013 November 18, 2013 dx.doi.org/10.1021/es404132w | Environ. Sci. Technol. 2013, 47, 14367−14375
Environmental Science & Technology
Article
(with 1% FBS) at the density of 5 × 105 cells/mL for 24 h at room temperature. The conditioned medium thus containing 1% FBS as well as secreted proteins was aspirated and purified as described but without diafiltration. The conditioned medium was stored frozen at −20 °C until use (within a few weeks). Nanoparticle−Protein Complexes. Nanoparticles. BioPure spherical 75 nm AgNPs (citrate-capped) were purchased from nanoComposix (San Diego, CA, USA). These AgNPs meet the OECD standards and are endotoxin-free (
Species Differences Take Shape at Nanoparticles: Protein Corona Made of the Native Repertoire Assists Cellular Interaction Yuya Hayashi,*,†,‡ Teodora Miclaus,† Carsten Scavenius,†,§ Katarzyna Kwiatkowska,∥ Andrzej Sobota,∥ Péter Engelmann,⊥ Janeck J. Scott-Fordsmand,‡ Jan J. Enghild,†,§ and Duncan S. Sutherland*,† †
iNANO Interdisciplinary Nanoscience Center, Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus C, Denmark Department of Bioscience-Soil Fauna Ecology and Ecotoxicology, Aarhus University, Vejlsøvej 25, 8600 Silkeborg, Denmark § Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10, 8000 Aarhus C, Denmark ∥ Department of Cell Biology, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland ⊥ Department of Immunology and Biotechnology, Clinical Center, University of Pécs, Szigeti u. 12, Pécs H-7643, Hungary ‡
S Supporting Information *
ABSTRACT: Cells recognize the biomolecular corona around a nanoparticle, but the biological identity of the complex may be considerably different among various species. This study explores the importance of protein corona composition for nanoparticle recognition by coelomocytes of the earthworm Eisenia fetida using E. fetida coelomic proteins (EfCP) as a native repertoire and fetal bovine serum (FBS) as a non-native reference. We have profiled proteins forming the long-lived corona around silver nanoparticles (75 nm OECD reference materials) and compared the responses of coelomocytes to protein coronas preformed of EfCP or FBS. We find that over time silver nanoparticles can competitively acquire a biological identity native to the cells in situ even in non-native media, and significantly greater cellular accumulation of the nanoparticles was observed with corona complexes preformed of EfCP (p < 0.05). An EfCP-nanoparticle mimicry made with a recombinant protein, lysenin, revealed its critical contribution in the observed cell−nanoparticle response. This confirms the determinant role of the recognizable biological identity during invertebrate in vitro testing of nanoparticles. Our finding shows a case of species-specific formation of biomolecular coronas, and this suggests that the use of representative species may need careful consideration in assessing the risks associated with nanoparticles.
■
INTRODUCTION Nanoparticles undergo considerable environmental transformations before reaching the target biological system,1 but their behavior and fate within the organism are then dictated by their biological identitythe biomolecular corona around nanoparticles (extensively reviewed in refs 2 and 3). Nature has diversified protein repertoires through evolution,4 and thereby the biological identity of nanoparticle−protein complexes may be considerably different among various species where there is little evolutionary conservation in the repertoire. Species differences of the nanoparticle’s biological identity could thus be translated into unique entities, each potentially having different toxicokinetics even among related species sharing similar ecological niches. Recent studies have characterized the formation of biomolecular coronas following incubation of different types of nanoparticles in fetal bovine serum (FBS) and human blood plasma (for a review see ref 2). Proteins interacting at nanoparticles may, for example, suffer conformational changes5,6 with resultant alteration of the biological functioning7,8 or initiation of inflammatory signaling.9 Nanoparticle− © 2013 American Chemical Society
protein complexes can acquire a differential biological identity and alter cell responses simply by whether the serum supplemented has been heat-inactivated,10 even with respect to the composition of cell culture media.11 In contrast, no studies exist that have explored the interaction of nanoparticles with proteins of invertebrate origins, although model invertebrate animals have been increasingly applied to in vitro settings12−14 as an integral part of nanomaterials ecotoxicology studies assessing at multilevels of biological organization.15−18 It is clear that adoption of mammalian in vitro practice creates an artificial biological milieu, in which the cells of invertebrates encounter exotic proteins of vertebrates and, most importantly, coronas of those extraneous proteins around nanoparticles. We have previously investigated fundamental aspects of how silver nanoparticles (AgNPs) bring about stress and immune responses that may be deeply conserved despite the evoluReceived: Revised: Accepted: Published: 14367
September 17, 2013 November 15, 2013 November 18, 2013 November 18, 2013 dx.doi.org/10.1021/es404132w | Environ. Sci. Technol. 2013, 47, 14367−14375
Environmental Science & Technology
Article
(with 1% FBS) at the density of 5 × 105 cells/mL for 24 h at room temperature. The conditioned medium thus containing 1% FBS as well as secreted proteins was aspirated and purified as described but without diafiltration. The conditioned medium was stored frozen at −20 °C until use (within a few weeks). Nanoparticle−Protein Complexes. Nanoparticles. BioPure spherical 75 nm AgNPs (citrate-capped) were purchased from nanoComposix (San Diego, CA, USA). These AgNPs meet the OECD standards and are endotoxin-free (
